Rolling Circle Amplification Tailored for Plasmonic Biosensors: From Ensemble to Single-Molecule Detection

We report on the tailoring of rolling circle amplification (RCA) for affinity biosensors relying on the optical probing of their surface with confined surface plasmon field. Affinity capture of the target analyte at the metallic sensor surface (e.g., by using immunoassays) is followed by the RCA step for subsequent readout based on increased refractive index (surface plasmon resonance, SPR) or RCA-incorporated high number of fluorophores (in surface plasmon-enhanced fluorescence, PEF). By combining SPR and PEF methods, this work investigates the impact of the conformation of long RCA-generated single-stranded DNA (ssDNA) chains to the plasmonic sensor response enhancement. In order to confine the RCA reaction within the evanescent surface plasmon field and hence maximize the sensor response, an interface carrying analyte-capturing molecules and additional guiding ssDNA strands (complementary to the repeating segments of RCA-generated chains) is developed. When using the circular padlock probe as a model target analyte, the PEF readout shows that the reported RCA implementation improves the limit of detection (LOD) from 13 pM to high femtomolar concentration when compared to direct labeling. The respective enhancement factor is of about 2 orders of magnitude, which agrees with the maximum number of fluorophore emitters attached to the RCA chain that is folded in the evanescent surface plasmon field by the developed biointerface. Moreover, the RCA allows facile visualizing of individual binding events by fluorescence microscopy, which enables direct counting of captured molecules. This approach offers a versatile route toward a fast digital readout format of single-molecule detection with further reduced LOD.

1. Overview of assays utilizing RCA-based amplification of output signal RCA increasingly serves as an amplification method in assays that rely on the affinity capture of target analyte species on a solid surface of a physico-chemical transducer. Table S1 provides selected examples of the optical, microbalance, magnetic, and electrochemical methods employed for the RCA-amplified assay readout with information on the medical diagnostics related application and achieved analytical performance (in terms of limit of detection -LOD). Table S1. Examples of RCA-based assays with information on the transducing mechanism, application, and achieved limit of detection. 2. Calibration of the surface density of anchoring points The circular padlock probe PL was prepared according to the description given in 'Methods' section of the manuscript. After the ligation and exonuclease reaction, the obtained circular PL was diluted to a given concentration c PL and ex-situ labelled by reacting with Cy5-LS* for 10 minutes before it was contacted with the pre-functionalized sensor surface carrying biotin-CS* probes. Solutions with increasing molar concentration of PL/Cy5-LS* complex (c = 4 pM to 40 nM) were flowed over the sensor chip surface and the fluorescence signal ΔF(t) was recorded by the PEF method. The fluorescence response ΔF was determined after the rinsing with working buffer (difference in the fluorescence intensity before and after the affinity binding) as the function of the molar concentration c and, as shown in Figure S1a, it saturates at c > 10 nM and exhibits Langmuir isotherm behavior. For the highest concentration of c = 40 nM, the surface mass density of the affinity captured padlock PL of ΔΓ = 0.18 ng/mm² was determined (as it induced a detectable shift in SPR) and it was related to the respective fluorescence response ΔF . By such determined conversion factor ΔF/ΔΓ, the surface mass density increase was extrapolated for all other lower concentrations c and average distance between the tethering points was calculated based on the known PL molecular weight as , (see Figure S1b). = • Δ

Affinity guiding of ssDNA chains on the surface
The guiding of the RCA-generated long ssDNA strands by affinity interaction of repeating chain segments with the sensor surface was first tested with a guiding sequence constructed of a biotin group, a 20-nucleotide thymine spacer and 32 sequence of GS strands. Interestingly, the RCA was then quenched and therefore the guiding sequence GS was shortened to 11 nucleotides (see Table 1 in the manuscript) in order to allow for weaker reversible attachment of RCA product. Then, the RCA process was not impaired and pronounced shift of SPR in the angular reflectivity curve occurs after the reaction (as can be seen in Figure S2a.). Additionally, two sharp dips at the location of the critical angle occur in the reflectivity spectrum R(θ), which indicate the production of a thick ssDNA layer supporting two dielectric waveguide modes. The surface mass density of the RCA chains was then obtained by fitting R(θ) with a Fresnel multilayer model. The determined refractive index n p and Figure S1. a) Fluorescence intensity ΔF measured for pre-labelled padlock probe with molar concentrations ranging from c PL = 40 pM to 40 nM correlated , b) relation between the molar concentration of the padlock probe c PL reacted with the sensor surface with the calculated average distance between the tethering points D.
S3 thickness d p translates to high increase in surface mass density of ΔΓ = 98.27 ng/mm 2 and was accompanied with strong fluorescence response ∆F = 5.94·10 4 cps was extracted from the angular fluorescence scans F(θ) for the excitation via surface plasmon waves.
Exposing the ssDNA to solutions with Ca 2+ (c = 10 mM) led additionally enhanced fluorescence signal ∆F in the low-density regime for D = 320 nm by a factor of 10.48, as shown in Figure S2b (red bars) on the interface carrying the 11-mer GS sequences. In the control experiment with randomized rGS strands, this strong enhancement is not observed. For the denser structure with D < 100 nm, the repulsion between tightly packed chains probably prevented the affinity binding of the RCA-strands to the guiding sequences GS, hence no enhancement effect was measured.

Fluorescence microscopy
The fluorescence images were acquired by confocal fluorescence microscopy after the RCA reaction and labeling with Cy5-LS. They were processed by the ImageJ software in order to determine the number of attached ssDNA chains, manifesting themselves as individual bright spots. By using a threshold, binary image was defined with identified high intensity area ( Figure S3) that were subsequently counted (spots exhibited area smaller than 10 µm² Figure S3b). The average distance between the spots were determined as square root of the whole image area divided by the number of counted spots. Figure S3. a) Analysis by the ImageJ software for a) determining the color threshold and b) counting the number of spots, shown for a plasmonic sensor chip with RCA-generated ssDNA from c = 400 fM. Figure S2. a) Angular reflectivity R(θ) and fluorescence scans F(θ) of a sensor surface reacted first with biotin-CS* and biotin-GS in a 1:1 ratio (red), subsequently binding of directly labeled PL with Cy5-LS* (black) and after one hour of RCA reaction, ssDNA reacted to Cy5-LS (blue), b) change of the fluorescence signal of Cy5-LS labeled ssDNA represented as ratio of ∆F when the strands are exposed to CaCl 2 and in PBST for the biointerface with the specific guiding sequence GS (red bars) and the random guiding sequence (grey bars).

Gel-electrophoresis
The 0.8% agarose gel electrophoresis was performed to prove the presence of RCA product in solution (not on the surface) after the reaction time of t = 30 s, 1 min, 2 min, 5 min, 10 min, 20 min. The RCA reaction mix contained 20 µL of circularized padlock probe with a molar concentration of 40 nM, 2 µL of biotin/20T/TSwith the molar concentration of 40 nM as primer, 1 µL of dNTPs with molar concentration of 25 µM and 20 Units of φ29-Polymerase with 13 µL NFW-BSA (0.2 mg/mL). The samples were incubated on the HulaMixer for the indicated time at room temperature and inactivated on the thermomixer at 70°C and 700 rpm for 10 minutes. Afterwards, 10 µL of the reaction mixture with 10 µL of 1:10 diluted loading dye were loaded in the wells of the agarose-gel and separated according to size at 100 V for about 30 to 40 minutes.
As shown in Figure S4a. series of dark bands becomes visible, located at higher kb-values with increasing reaction time t, which can be associated with the RCA-generated ssDNA. From the width of the bands a distribution of the RCA-generated DNA length can be estimated, also reported in previous publications. For longer RCA-times, the length of ssDNA exceeds the range that can be quantified by using the used ladder (shown on the left side). The speed of prolongation when the RCA is carried out in the bulk solution can be estimated above 10 3 nt/min, which is higher than 215 nt/min determined on the surface in our previous work. 12 Figure S4b shows the RCA reaction in solution after 10 min in comparison with the padlock probe (c = 40 nM).
As the circular DNA probe has only 81 nt, the dark band is located at lower bp values when compared to the 100 bp ladder. The intensity of the dark band is higher for the RCA product which can be associated to a high amount of long ssDNA strands.

Atomic force microscopy
The RCA protocol was carried out on the SPR sensor chips surface in the flow cell for the same conditions as used for the optical measurement. The assay was carried out for the concentration of the padlock probe of 400 fM. Figure S5 shows the topography acquired by the AFM (see methods part in the manuscript) for the surface without and with the specific guiding sequences immobilized.